Image pro plus software

Immunohistochemistry has been applied for determining where the FIZZ1 protein was localized and its concentration. Lung paraffin sections were deparaffinized, rehydrated, and blocked by 3% H₂O₂. Sections were then blocked with goat serum at a concentration of 5% to reduce the absorption of non-specific immunoglobulins. After that, the specimens were incubated with anti-FIZZ1 antibody (1:1,000, ab39626, Abcam) at 37°C for 2 h, followed by incubating with a secondary antibody at 37°C for 30 min. Image-Pro Plus software (Version X; Adobe, San Jose, CA) has been applied for evaluating protein expression. In total, eight images of bronchioles from each tissue section were examined using ×400 magnification and the program Case Viewer (version 1.3; 3DHistech, Budapest, Hungary). Data were presented as an average optical density (AOD) for the entire sections of lung tissue showing positive staining.

For the immunofluorescence assay, the lung sections were treated with rabbit anti-RELM-β polyclonal antibodies (1:200, Abcam, UK) and mouse anti-α-SMA antibody (1:500) or the corresponding vehicle overnight at 4 °C. Then, the sections were incubated with FITC-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:50, Boster, CHN) (excitation: 495 nm, emission: 525 nm) and Cy3-conjugated AffiniPure goat anti-mouse IgG (H + L) (1:50, Boster, CHN) (excitation: 554 nm, emission: 568 nm) for 45 min at room temperature in the dark. Finally, the sections were washed in PBS and mounted with Vectashield hardset mounting medium with 4′,6-diamidino-2-phenylindole dilactate (DAPI, Vector Laboratories, USA). Specimens were observed under a fluorescent inverted microscope (IX73-A22FL/PH; Olympus Corporation; light source: UHP) attached to a CCD digital camera (512B Cascade, Roper Scientific, Tucson, AZ). Blue (WU) and green filters (WIBA) were used to detect FITC (green) and Cy3 (red) signals, respectively. The objectives used were 20 × and 40 × . Images were captured by using the Image-Pro Plus software (version 6.0) and colorized with Adobe Photoshop CS6 software (Adobe Systems, Inc., San Jose, CA).

After micro‐CT scanning, the specimens were dehydrated in a graded ethanol series (70–100%) and then embedded in a methylmethacrylate solution that was polymerized at 37 °C within 1 week. Afterward, thin sections (≈50 µm in thickness) were prepared using the modified interlocked diamond saw (Leica Microtome, Wetzlar, Germany) and stained with 1.2% trinitrophenol and 1% acid fuchsin (Van‐Gieson staining). Bone formation was qualitatively measured with a standard light microscope (Leica) equipped with a digital image analysis system (Image‐Pro Plus software, Media Cybernetics, Silver Spring, USA). Prior to histomorphometric analysis, bone and material were pseudocoloured using Adobe Photoshop 6.0 and then measured using a digital image analysis system (Image‐Pro Plus software, Silver Spring, USA). The bone volume fraction (ratio of bone tissue area to the implant pore area within the implant) was calculated based on Van‐Gieson staining and compared statistically.

Thioflavin-S (Th-S), a fluorescent dye, is used as part of a common method for staining senile plaques [35 (link)]. Brain tissue of mice was first fixed with paraformaldehyde and then dehydrated and embedded in paraffin. Tissues embedded in paraffin were sectioned with a microtome. Four micrometer paraffin brain sections were collected on slides. Th-S staining was carried out in sections as previously described [36 (link), 37 ]. Sections were dehydrated and rehydrated in distilled water and then stained with Mayer's hematoxylin for 5 min. Then, they were rinsed with running water for 1 min and immersed in the Th-S solution (1% Th-S in distilled water) for 5 min. Slices were immersed in 70% alcohol for 5 min and washed with distilled water 2 times. Glycerol gelatin was used to hold the coverslips. All slices were observed by another investigator who was blinded using an optical microscope (Olympus BX 41 microscope, 40x magnification). Pictures of each group (n ≥ 10) were further processed using Image-Pro Plus software (version 5.0) and Photoshop software (Adobe Systems). The results are presented as the mean area of the Th-S positive Aβ deposits counts in a total field area of 1.6 mm × 1.6 mm.